We are using the tryptophan synthase multienzyme alpha-2-beta-2 complex as a model system for investigating how enzymes interact, communicate, and channel reaction intermediates. Our previous X-ray crystallographic studies of the wild type tryptophan synthase alpha-2-beta-2 complex from Salmonella typhimurium reveal that the active sites of the alpha and beta subunits are 25 angstroms apart and are connected by a tunnel. We are using the crystal structure as the basis for engineering mutations designed to alter the tunnel and to alter subunit interaction and communication. During the past year we have used several approaches to analyze wild type and mutant enzymes. (1) Alterations in the tunnel at positions 170 and 280 of the beta subunit have been investigated by analyses with a fluorescent probe (Nile Red), by rapid kinetic studies, and by X-ray crystallography. Replacing beta Cys-170 by a bulky tryptophan results in partial or total blocking of the tunnel. Our results indicate that beta site ligands and alpha site ligands convert the tunnel from an open form to a closed form. (2) Studies of the effects of alcohol cosolvents on the spectroscopic and catalytic properties of the beta subunit in the alpha-2-beta-2 complex show that cosolvents stabilize the open form of the beta subunit in the alpha-2- beta-2 complex. (3) X-ray crystallographic studies have refined the structures of the wild type alpha-2-beta-2 complex and of Schiff base intermediates of a mutant alpha-2-beta-2 complex (K87T) to about 2 angstroms resolution. The results reveal ligand-induced conformational changes. (4) The allosteric activation of the alpha subunit in the alpha-2-beta-2 complex by a beta site ligand, L-serine, has been analyzed by steady-state and rapid kinetic studies using an alpha-2-beta-2 complex with a mutation in the beta subunit (k87T). The results provide evidence that the Schiff base of aminoacrylate is the key intermediate at the beta site that triggers activation of the alpha subunit in the alpha-2-beta-2 complex. (5) Thermally-induced unfolding of the separate alpha and beta subunits and alpha-2-beta-2 complex has been analyzed by several techniques: UV-visible and circular dichroism spectroscopy, activity measurements and differential scanning calorimetry. The stepwise unfolding of the beta subunit is modulated by the presence of the alpha subunit. These results permit correlation of thermodynamic and structural information.